This invention relates to a chimaeric human papillomavirus (HPV) virus like particle (VLP) having a diameter of about 30 nm and a method of treatment and/or prophylaxis of HPV infection and/or cervical cancer by administration of the chimaeric HPV VLP of the invention.
Cervical cancer is primarily caused by HPV infection and is the third most common cancer among women worldwide (Ferlay et al., 2010). As a result, HPV vaccine development is a priority for preventative cancer research. The L1 major capsid protein is the antigen of choice for prophylactic vaccines, as it is immunodominant and self-assembles into VLPs which are structurally and immunologically similar to authentic virions. Vaccination with VLPs elicits high titres of neutralisation antibodies (NAb) in both animals and humans and two multivalent HPV L1 VLP-based prophylactic vaccines have been licensed and are highly effective in the prevention of vaccine-type HPV-16 and 18 infections and associated disease (Schiller et al., 2008).
Despite the high efficacy of current L1 VLP-based HPV vaccines, the type-specificity (Brown et al., 2009; Wheeler et al., 2009), the lack of therapeutic efficacy (FUTURE II Study Group, 2007; Hildersheim et al., 2007) and the high cost of vaccines (Schiller et al., 2008) have limited their widespread application, particularly in developing countries with >80% of the cervical cancer burden (Parkin and Bray, 2006). Therefore, there is an urgent need for affordable second generation HPV vaccines, which broaden protection to include multiple oncogenic HPV types, and improve the therapeutic efficacy to clear established HPV infections and cancerous lesions.
Broad-spectrum prophylactic HPV vaccines can be developed using cross neutralising L2 epitopes. The L2 epitopes can be incorporated into surface regions of L1 to create L1/L2 chimaeras displaying the L2 peptide on the surface of assembled L1 (WO 03/097673; Kawana et al., 1999, 2003; Slupetzky et al., 2007; Kondo et al., 2007, 2008).
The use of plant expression systems for the large-scale production of foreign antigens has been proposed as a cost-effective alternative for vaccine production (Fischer et al., 2004), with a definitive trend toward the use of transient expression for high-level protein expression and optimisation (Rybicki, 2009). Several groups have expressed HPV-16 L1 in plants (Biemelt et al., 2003; WO 2006/119516; Maclean et al., 2007).
A practical limitation of plant systems is low yields of recombinant protein, potentially a result of protein instability or low-level expression (Fischer et al., 2004; Obembe et al., 2011). It is estimated that plant-expressed recombinant protein yields need to be greater than 1% of the total soluble protein (TSP) to be economically viable (Fischer et al., 2004). This is particularly problematic for the expression of recombinant proteins using nuclear-transformed transgenic plants, as these systems are often associated with low yields of recombinant protein (Rybicki, 2009).
HPV-16 L1 has been expressed transgenicaily in nuclear-transformed potato and tobacco plants, but low expression levels of HPV-16 L1 (<1% TSP) have consistently reported and the elicited immune responses were relatively weak (Biemelt et al., 2003; Varsani et al., 2003b; Varsani et al., 2006a).
However, human codon-optimisation of the L1 gene and targeting to the chloroplast have significantly improved HPV-16 L1 expression in both transgenic and Agrobacterium-mediated transient tobacco expression systems to up to about 17% TSP (Maclean et al., 2007).
A recent development in plant-derived HPV vaccines was the expression of the first HPV-16 L1 chimaera in plants. The L1/E6/E7 chimaera consisted of HPV-16 L1 C-terminally fused to several E6 and E7 epitopes and it was expressed in transgenic tomatoes (Paz De la Rosa et al., 2009). However, yields were low (0.05-0.1% TSP) and therefore not commercially viable.
WO 2011/077371 describes a method for producing chimaeric HPV L1 polypeptides with increased expression levels relative to HPV L1 protein in an insect, plant or yeast expression system. Although human codon-optimised L1/L2 chimaeras produced from HPV L1 and BPV L2 (amino acids 1-88) in plants formed VLPs of about 55 nm, the other HPV L1/L2 chimaeras were only able to form capsomeres of approximately 17 nm in diameter.
Although capsomeres are stable at room temperature, they are only able to induce 20 to 40-fold lower humoral immune responses in comparison to VLPs (Thönes et al., 2008). It would therefore be beneficial to develop a chimaeric VLP comprising L1 and L2 which is expressed at commercially viable levels in an expression system. Such a chimaeric VLP would be easier to purify and is likely to be more immunogenic than a chimaeric capsomere.